Method for analyzing by-products of rna in vitro transcription

ABSTRACT

The present invention relates to the detection and analysis of by-products in a process of RNA in vitro transcription by HPLC. It further relates to the use of this method for the quality control of RNA produced by in vitro transcription or for identifying suitable RNA purification conditions.

FIELD OF THE INVENTION

The present invention relates to the detection and analysis ofby-products in a process of RNA in vitro transcription by HPLC. Itfurther relates to the use of this method for the quality control of RNAproduced by in vitro transcription or for identifying suitable RNApurification conditions.

BACKGROUND OF THE INVENTION

For the therapeutic use of RNA in patients, a rigorous quality controlof the RNAs to be used is mandatory. An important issue is thedetermination of RNA purity. Apart from RNA integrity, which is commonlydetermined via gel electrophoresis, limited knowledge is available as towhich parameters are important for RNA quality.

During transcription, RNAs shorter than the target RNA are also producedby the polymerase. These may alter the properties of the mRNA product,not only in terms of concentration, but also in terms of biologicalactivity, if not thoroughly removed.

It is well established that RNA transcribed in vitro by phage polymerasecontains multiple aberrant RNAs, including short RNAs as a result ofabortive transcription initiation events (Milligan et al. (1987) Nucl.Acids Res 15:8783-8798) and double stranded (ds)RNAs generated by RNAdependent RNA polymerase activity (Arnaud-Barbe et al. (1998) Nucl.Acids Res 26:3550-3554; Nacheva and Berzal-Herranz (2003) Eur. J.Biochem. 270:1458-1465).

Kariko et al. identified that these contaminants from in vitrotranscribed RNA are a source of innate immune activation and that theirremoval increases RNA translation and eliminates type I interferon andinflammatory cytokine secretion (Karikó K. et al. (2011) Nucl. AcidsRes. 39(21):e142)

These short RNAs cannot be detected in standard gel electrophoresisanalysis of long mRNAs wherein RNA is visualized by intercalating dyessuch as ethidium bromide. These dyes intercalate and/or interact withthe phosphate backbone, resulting in good visualization of longernucleic acids, whereas short RNAs are difficult to detect, especiallywhen present in a mixture with longer RNAs, such as mRNAs. Additionally,the methods used to resolve long RNAs cannot be used to resolve shortRNAs.

For the future development of RNA products/medicaments it is mandatoryto develop a method for determining the presence and quantity of shortRNA by-products as a quality control.

WO 2015/101416 A1 and PCT/EP2015/001336 describe methods for analyzingan RNA molecule wherein the RNA molecule is cleaved with a catalyticnucleic acid molecule and the resulting RNA fragments are analyzed.

WO 2014/144039 A1 describes a method for characterizing an RNAtranscript using a procedure selected from the group consisting ofoligonucleotide mapping, reverse transcriptase sequencing, chargedistribution analysis and detection of RNA impurities.

Hence, the problem of the invention is to provide a sensitive method forreliably detecting by-products of RNA in vitro transcription.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that short by-products ofRNA in vitro transcription can be detected and analyzed by HPLC. An HPLCprotocol has been developed that allows single-nucleotide resolution ofRNA oligomers for monitoring and analysis of transcription reactions andRNA products. Using this protocol, contamination of the RNA product withby-products can be determined and quantified. Additionally, fractioncollection of selected peaks during HPLC purification allows theisolation and subsequent characterization of the RNA species comprisedin the by-products for identifying crucial sequence motifs responsiblefor the generation of by-products and for identifying betterpurification methods to improve RNA quality.

Accordingly, the present invention relates to a method for detectingby-products of in vitro transcription in a sample comprising an in vitrotranscribed target RNA, the method comprising the steps of:

-   -   a) preparing a sample comprising a target RNA by in vitro        transcription;    -   b) purifying the target RNA, thereby providing a purified target        RNA sample;    -   c) detecting the by-products in the purified target RNA sample        by HPLC.

Preferably, the method does not comprise a step of treating the targetRNA with a ribozyme.

The by-products may comprise at least two nucleic acid molecules withdifferent length and may have a length of 5 to 500 nucleotides.

Preferably, the by-products do not comprise the 3′ terminus of thetarget RNA. The by-products may be homooligomers of nucleotides, shortsingle-stranded RNAs, double-stranded RNAs and/or DNA-RNA hybrids.

Step b) may be performed under denaturing conditions and/or may comprisea step of purifying the target RNA by HPLC, which preferably isreversed-phase HPLC.

In the HPLC a porous reversed phase may be used as stationary phase,which preferably is a porous, non-alkylated polystyrene/divinylbenzenematrix.

The HPLC in step c) may be ion-pair, reversed-phase HPLC and/or may usea carbon-chain bonded silica column.

Preferably, the carbon-chain bonded silica column is an octadecyl carbonchain (C18)-bonded silica column.

The silica column may be prepared from tetraethoxysilane andbis(triethoxysilyl)ethane which may be used in a 4:1 mole ratio.

In one embodiment the column has a particle size of 0.5 to 5 μm and/orhas a pore size of 50 to 300 Å.

The HPLC of step c) may use a mixture of an aqueous solvent and anorganic solvent as mobile phase.

The aqueous solvent may be a buffer which may be selected from the groupconsisting of triethylammonium acetate, trifluoroacetic acid, aceticacid, formic acid, acetate buffer, phosphate buffer, tetrabutylammoniumbisulfate, tetrabutylammonium bromide and tetrabutylammonium chloride.

Preferably, the buffer is a 0.1 M triethylammonium acetate buffer.

The organic solvent may be selected from the group consisting ofacetonitrile, methanol, ethanol, 1-propanol, 2-propanol,hexafluoroisopropanol, acetone and a mixture thereof and preferably itmay be acetonitrile.

In one embodiment the mobile phase contains 3 to 5% organic solvent,relative to the mobile phase, the rest being the aqueous solvent at thebeginning of the HPLC process.

In one embodiment a gradient separation proceeds and preferably theproportion of organic solvent is increased to provide the gradient. Morepreferably, the proportion of organic solvent in the mobile phase isincreased in the course of HPLC separation from 3.5% to 100%.

The method may further comprise a step d) of isolating and/orcharacterizing the by-products.

The by-products may be characterized by enzyme assays, mass spectrometryand/or sequencing.

In one embodiment the amount of the by-products relative to the totalamount of RNA is determined.

The method of the present invention may be used to identify sequencemotifs within the target RNA which are responsible for the generation ofby-products.

The method of the present invention may also be used for the qualitycontrol of RNA produced by in vitro transcription.

The method of the present invention may also be used to identifysuitable RNA purification conditions.

The method of the present invention may also be used to compare RNApurification conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: By-products of in vitro transcription of PpLuc mRNA (R491 (lightgrey), R1265 (dark grey), and R2244 (black)) detected via analyticalHPLC (C18 column).

(a) Chromatogram for entire analytical run. Full-length mRNA elutes ataround 24 min.(b) Enlargement of the area between 1 and 23 min of the chromatogram of(a). Short RNAs are resolved by single nucleotides. Three distinctpopulations of short RNAs have been identified:

-   -   “early peaks” (1), whose generation is dependent upon the mRNA        start sequence (occurring only in R2244) and which consist of        guanosine multimers (as determined by mass spectrometry);    -   “middle peaks” (2), which are antisense RNA oligomers        transcribed via RNA-dependent RNA polymerization (as determined        by mass spectrometry), in this case from the open reading frame        sequence of PpLuc;    -   “late peaks” (3).

FIG. 2: HPLC analysis of PpLuc mRNA (R491 (light grey), R1265 (darkgrey), and R2244 (black)) previously purified via an improvedpreparative HPLC method. Hybridized RNA oligomers could be removed,resulting in high quality RNA.

(a) Chromatogram for entire analytical run. Full-length mRNA elutes ataround 24 min.(b) Enlargement of the area between 1 and 23 min of the chromatogram of(a) (same scaling as in FIG. (1 b)).

DEFINITIONS

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned in these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

In vitro transcription: The term “in vitro transcription” relates to aprocess wherein RNA is synthesized in a cell-free system (in vitro).DNA, particularly plasmid DNA, is used as template for the generation ofRNA transcripts. RNA may be obtained by DNA-dependent in vitrotranscription of an appropriate DNA template, which according to thepresent invention is preferably a linearized plasmid DNA template. Thepromoter for controlling in vitro transcription can be any promoter forany DNA-dependent RNA polymerase. Particular examples of DNA-dependentRNA polymerases are the T7, T3, and SP6 RNA polymerases. A DNA templatefor in vitro RNA transcription may be obtained by cloning of a nucleicacid, in particular cDNA corresponding to the respective RNA to be invitro transcribed, and introducing it into an appropriate vector for invitro transcription, for example into plasmid DNA. In a preferredembodiment of the present invention the DNA template is linearized witha suitable restriction enzyme, before it is transcribed in vitro. ThecDNA may be obtained by reverse transcription of mRNA or chemicalsynthesis. Moreover, the DNA template for in vitro RNA synthesis mayalso be obtained by gene synthesis.

Methods for in vitro transcription are known in the art (Geall et al.(2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) MethodsEnzymol. 530:101-14). Reagents used in said method typically include:

1) a linearized DNA template with a promoter sequence that has a highbinding affinity for its respective RNA polymerase such asbacteriophage-encoded RNA polymerases;2) ribonucleoside triphosphates (NTPs) for the four bases (adenine,cytosine, guanine and uracil);3) optionally a cap analog as defined below (e.g. m7G(5′)ppp(5′)G(m7G));4) a DNA-dependent RNA polymerase capable of binding to the promotersequence within the linearized DNA template (e.g. T7, T3 or SP6 RNApolymerase);5) optionally a ribonuclease (RNase) inhibitor to inactivate anycontaminating RNase;6) optionally a pyrophosphatase to degrade pyrophosphate, which mayinhibit transcription;7) MgCl₂, which supplies Mg²⁺ ions as a co-factor for the polymerase;8) a buffer to maintain a suitable pH value, which can also containantioxidants (e.g. DTT), and/or polyamines such as spermidine at optimalconcentrations.

By-product: A by-product is a secondary product of a manufacturingprocess or a chemical reaction, which differs from the target product ofsaid process or reaction in its size and/or chemical structure. Withinthe present invention the by-product is produced by the RNA polymeraseduring the RNA in vitro transcription process.

Within the present invention the by-product may comprise homooligo- or-polymers of a nucleotide, such as oligomers of guanosine, for exampleoligomers comprising 3 to 10 guanosine nucleotides.

Additionally or alternatively, the by-product may comprise short RNAswhich have a lower number of nucleotides than the target RNA, but havepart of the sequence of the target RNA and may therefore also beconsidered as fragments of the target RNA. These short RNAs may forexample be produced by premature termination of transcription, i.e. thetranscription stops before the end of the sequence to be transcribed isreached. Hence, these short RNAs typically comprise the 5′ sequence ofthe target RNA.

Also additionally or alternatively, the by-product may comprise longRNAs which have a higher number of nucleotides than the target RNA andcomprise the complete sequence of the target RNA and additionalnucleotides. These long RNAs may for example be produced by incompletetermination of the transcription or by incomplete linearization of theplasmid providing the template DNA.

Also additionally or alternatively, the by-products may comprisedouble-stranded RNA or DNA/RNA hybrids which are produced byRNA-dependent polymerization catalyzed by the RNA polymerase. In themethod of the present invention the detection of antisense RNA or DNAmolecules may be indicative for these by-products.

The by-product may also be an RNA having the same or a shorter or longerlength as the target RNA in which one or more modified nucleotides arepresent, if the target RNA does not comprise modified nucleotides.

Nucleic acid: The term nucleic acid means any DNA- or RNA-molecule andis used synonymously with polynucleotide. Furthermore, modifications orderivatives of the nucleic acid as defined herein are explicitlyincluded in the general term “nucleic acid”. For example, peptidenucleic acid (PNA) is also included in the term “nucleic acid”.

Target RNA: The target RNA is the RNA which is to be produced by the RNAin vitro transcription process. The length and the sequence of thetarget RNA is determined by the sequence of the nucleic acid templatewhich is subjected to the RNA in vitro transcription reaction. Hence,the target RNA is the full-length RNA transcript. In contrast, theby-products typically are either longer or shorter than the target RNA.The target RNA may further comprise a cap structure on its 5′ terminus,if a cap analog is added to the RNA in vitro transcription reaction. Thetarget RNA may also comprise modified nucleotides, if these modifiednucleotides had been added to the RNA in vitro transcription reactionmixture. In contrast, RNA containing modified nucleotides which had notbeen added to the RNA in vitro transcription reaction mixture isconsidered as a by-product.

If the target RNA is mRNA, it will preferably code for proteins, inparticular those which have an antigen character, and for example allrecombinantly produced or naturally occurring proteins, which are knownto a person skilled in the art from the prior art and are used fortherapeutic, diagnostic or research purposes. In particular, theantigens may be tumour antigens or antigens of pathogens, for example ofviral, bacterial or protozoal organisms.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.These nucleotides are usually adenosine-monophosphate (AMP),uridine-monophosphate (UMP), guanosine-monophosphate (GMP) andcytidine-monophosphate (CMP) monomers or analogs thereof, which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificorder of the monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the RNA sequence. Usually RNA may beobtainable by transcription of a DNA sequence, e.g., inside a cell. Ineukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. In vivo, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap,optionally a 5′UTR, an open reading frame, optionally a 3′UTR and apoly(A) sequence.

In addition to messenger RNA, several non-coding types of RNA existwhich may be involved in regulation of transcription and/or translation,and immunostimulation.

The term “RNA” further encompasses RNA molecules, such as viral RNA,retroviral RNA and replicon RNA, small interfering RNA (siRNA),antisense RNA, CRISPR RNA, ribozymes, aptamers, riboswitches,immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), smallnuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), andPiwi-interacting RNA (piRNA).

Modified nucleoside triphosphate: The term “modified nucleosidetriphosphate” as used herein refers to chemical modifications comprisingbackbone modifications as well as sugar modifications or basemodifications. These modified nucleoside triphosphates are herein alsocalled (nucleotide) analogs.

In this context, the modified nucleoside triphosphates as defined hereinare nucleotide analogs/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides are chemically modified. Asugar modification in connection with the present invention is achemical modification of the sugar of the nucleotides. Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides. In this contextnucleotide analogs or modifications are preferably selected fromnucleotide analogs which are applicable for transcription and/ortranslation.

Sugar Modifications

The modified nucleosides and nucleotides, which may be used in thecontext of the present invention, can be modified in the sugar moiety.For example, the 2′ hydroxyl group (OH) can be modified or replaced witha number of different “oxy” or “deoxy” substituents. Examples of“oxy”-2′ hydroxyl group modifications include, but are not limited to,alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar); polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR;“locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected,e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar;and amino groups (—O-amino, wherein the amino group, e.g., NRR, can bealkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) oraminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleotide can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides. The phosphate groups of the backbone can bemodified by replacing one or more of the oxygen atoms with a differentsubstituent. Further, the modified nucleosides and nucleotides caninclude the full replacement of an unmodified phosphate moiety with amodified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications

The modified nucleosides and nucleotides, which may be used in thepresent invention, can further be modified in the nucleobase moiety.Examples of nucleobases found in RNA include, but are not limited to,adenine, guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In some embodiments, the nucleotide analogs/modifications are selectedfrom base modifications, which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine,2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5 ‘-O-(1-Thiophosphate)-Cytidine,5’-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine.

In further specific embodiments the modified nucleotides includenucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine,7-deaza-adenosine.

Further modified nucleotides have been described previously (WO2013/052523).

5′-Cap structure: A 5′ cap is typically a modified nucleotide,particularly a guanine nucleotide, added to the 5′ end of an RNAmolecule. Preferably, the 5′ cap is added using a 5′-5′-triphosphatelinkage. A 5′ cap may be methylated, e.g. m7GpppN, wherein N is theterminal 5′ nucleotide of the nucleic acid carrying the 5′ cap,typically the 5′-end of an RNA. The naturally occurring 5′ cap ism7GpppN.

Further examples of 5′ cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety.

Particularly preferred 5′ cap structures are CAP1 (methylation of theribose of the adjacent nucleotide of m7G), CAP2 (methylation of theribose of the 2^(nd) nucleotide downstream of the m7G), CAP3(methylation of the ribose of the 3^(rd) nucleotide downstream of them7G) and CAP4 (methylation of the ribose of the 4^(th) nucleotidedownstream of the m7G).

A 5′ cap structure may be formed by a cap analog.

Cap analog: A cap analog refers to a non-extendable di-nucleotide thathas cap functionality which means that it facilitates translation orlocalization, and/or prevents degradation of the RNA molecule whenincorporated at the 5′ end of the RNA molecule. Non-extendable meansthat the cap analog will be incorporated only at the 5′terminus becauseit does not have a 5′ triphosphate and therefore cannot be extended inthe 3′ direction by a template-dependent RNA polymerase.

Cap analogs include, but are not limited to, a chemical structureselected from the group consisting of m7GpppG, m7GpppA, m7GpppC;unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g.,m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylatedsymmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs(e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG andtheir tetraphosphate derivatives) (Stepinski et al., 2001. RNA7(10):1486-95). Examples of cap analogs are shown in Table 1.

TABLE 1 Cap analogs (D1 and D2 denote counterpart diastereoisomers)Triphosphate cap analog Tetraphosphate cap analog m⁷Gp₃G m⁷Gp₄G m₂^(7,3′-O)Gp₃G b⁷Gp₄G b⁷Gp₃G b⁷m^(3′-O)Gp₄G e⁷Gp₃G m₂ ^(2,7)Gp₄G m₂^(2,7)Gp₃G m₃ ^(2,2,7)Gp₄G m₃ ^(2,2,7)Gp₃G b⁷m²Gp₄G m⁷Gp₃2′dG m7Gp⁴m⁷Gm⁷Gp₃m^(2′-O)G m⁷Gp₃m⁷G m₂ ^(7,2′-O)Gp₃G m₂ ^(7,2′-O)Gppp_(s)G (D1) m₂^(7,2′-O)Gppp_(s)G (D2) m₂ ^(7,2′-O)Gpp_(s)pG (D1) m₂ ^(7,2′-O)Gpp_(s)pG(D2) m₂ ^(7,2′-O)Gp_(s)ppG (D1) m₂ ^(7,2′-O)Gp_(s)ppG (D2)

Further cap analogs have been described previously (U.S. Pat. No.7,074,596, WO 2008/016473, WO 2008/157688, WO 2009/149253, WO2011/015347, and WO 2013/059475). The synthesis ofN⁷-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs has beendescribed recently (Kore et al., 2013. Bioorg. Med. Chem.21(15):4570-4).

Particularly preferred cap analogs are G[5′]ppp[5′]G, m⁷G[5′]ppp[5′]G,m₃ ^(2,2,7)G[5′]ppp[5′]G, m₂ ^(7,3′-O)G[5′]ppp[5′]G (3′-ARCA), m₂^(7,2′-O)GpppG (2′-ARCA), m₂ ^(72′-O)GppspG D1 (β-S-ARCA D1) and m₂^(7,2′-O)GppspG D2 (β-S-ARCA D2).

Purification/purifying: The terms “purification”, “purified” or“purifying” are intended to mean that the target RNA is separated and/orisolated from the by-products and the components of the RNA in vitrotranscription reaction present in the sample comprising the target RNAafter the RNA in vitro transcription reaction. Thus, after purificationthe purified target RNA sample has a higher purity than the targetRNA-containing sample prior to purification, i.e. the amount ofby-products and the components of the RNA in vitro transcriptionreaction is lower than in the sample after transcription, but beforepurification. Undesired constituents of RNA-containing samples whichtherefore need to be separated may in particular be by-products of theRNA in vitro transcription reaction, or also excessively longtranscripts if plasmids are not completely linearised. In addition,components of the RNA in vitro transcription reaction mixture, such asenzymes, for example RNases and polymerases, and nucleotides may beseparated from the target RNA in the purification step.

After the purification step, the target RNA has a higher purity thanbefore the purification step, but may still contain by-products whichmay be detected by the method of the present invention. The degree ofpurity after the purification step may be more than 70% or 75%, inparticular more than 80% or 85%, very particularly more than 90% or 95%and most favorably 99% or more. The degree of purity may for example bedetermined by an analytical HPLC as described herein, wherein thepercentage provided above corresponds to the ratio between the area ofthe peak for the target RNA and the total area of all peaks representingthe by-products.

HPLC: HPLC is the common abbreviation of the term “high performanceliquid chromatography”. In the HPLC process a pressurized liquid solventcontaining the sample mixture is passed through a column filled with asolid adsorbent material leading to the interaction of components of thesample with the adsorbent material. Since different components interactdifferently with the adsorbent material, this leads to the separation ofthe components as they flow out of the column. The operational pressurein HPLC process is typically between 50 and 350 bar. The term HPLCincludes reversed phase HPLC (RP-HPLC), size exclusion chromatography,gel filtration, affinity chromatography, hydrophobic interactionchromatography or ion pair chromatography, wherein reversed phase HPLCis preferred.

Reversed phase HPLC: Reversed phase HPLC uses a non-polar stationaryphase and a moderately polar mobile phase and therefore works withhydrophobic interactions which result from repulsive forces between arelatively polar solvent, the relatively non-polar analyte, and thenon-polar stationary phase (reversed phase principle). The retentiontime on the column is therefore longer for molecules which are morenon-polar in nature, allowing polar molecules to elute more readily. Theretention time is increased by the addition of polar solvent to themobile phase and decreased by the addition of more hydrophobic solvent.

The characteristics of the specific RNA molecule as an analyte may playan important role in its retention characteristics. In general, ananalyte having more apolar functional groups results in a longerretention time because it increases the molecule's hydrophobicity andtherefore the interaction with the non-polar stationary phase. Verylarge molecules, however, can result in incomplete interaction betweenthe large analyte surface and the alkyl chain. Retention time increaseswith hydrophobic surface area which is roughly inversely proportional tosolute size. Branched chain compounds elute more rapidly than theircorresponding isomers because the overall surface area is decreased.

Ion-pair, reversed-phase HPLC: Ion-pair, reversed-phase HPLC is aspecific form of reversed-phase HPLC in which an ion with a lipophilicresidue and positive charge such as an alkylammonium salt, e.g.triethylammonium acetate, is added to the mobile phase as counterion forthe negatively charged RNA. When used with common hydrophobic HPLCphases in the reversed-phase mode, ion pair reagents can be used toselectively increase the retention of the RNA.

Ribozyme: A ribozyme is a catalytic nucleic acid molecule which is anRNA molecule capable of catalyzing reactions including, but not limitedto, site-specific cleavage of other nucleic acid molecules such as RNAmolecules. The term ribozyme is used interchangeably with phrases suchas catalytic RNA, enzymatic RNA, or RNA enzyme.

Ribozymes are broadly grouped into two classes based on their size andreaction mechanisms: large and small ribozymes. The first group consistsof the self-splicing group I and group II introns as well as the RNAcomponent of RNase P, whereas the latter group includes the hammerhead,hairpin, hepatitis delta ribozymes and varkud satellite (VS) RNA as wellas artificially selected nucleic acids. Large ribozymes consist ofseveral hundred up to 3000 nucleotides and they generate reactionproducts with a free 3′-hydroxyl and 5′-phosphate group. In contrast,small catalytically active nucleic acids from 30 to −150 nucleotides inlength generate products with a 2′-3′-cyclic phosphate and a 5′-hydroxylgroup (Schubert and Kurreck (2004) Curr. Drug Targets 5(8):667-681).

3′ terminus of the target RNA: The 3′ terminus of the target RNA is aregion comprising nucleotides from the 3′ terminal part of the targetRNA. Hence, the 3′ terminus has the same sequence as the correspondingpart of the target RNA. The 3′ terminus comprises at least a part of thepoly(A) sequence which is the most 3′ part of the target RNA and mayadditionally comprise part of the open reading frame and/or optionallythe 3′ UTR (if it is encoded by the DNA template).

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention is based on the finding thatby-products of RNA in vitro transcription can be detected and analyzedby HPLC.

Accordingly, the present invention relates to a method for detectingby-products of in vitro transcription in a sample comprising an in vitrotranscribed target RNA, the method comprising the steps of:

-   -   a) preparing a sample comprising a target RNA by in vitro        transcription;    -   b) purifying the target RNA, thereby providing a purified target        RNA sample;    -   c) detecting the by-products in the purified target RNA sample        by HPLC.

In one embodiment the method does not comprise a step of treating thetarget RNA with a ribozyme, in particular a ribozyme which is designedto cleave within the target RNA. Hence, the method does not involve astep of intentionally cleaving the target RNA to create smallerfragments, but the by-products which are detected and optionallyanalyzed by the method of the present invention are createdunintentionally during the process of RNA in vitro transcription withoutrequiring any step of treating the target RNA to create smaller RNAfragments.

In this context it is particularly preferred that the present inventiondoes not relate to a method for analyzing an RNA molecule having acleavage site for a catalytic nucleic acid molecule or a method foranalyzing a population of RNA molecules, wherein the populationcomprises at least one RNA molecule that has a cleavage site for acatalytic nucleic acid molecule, comprising a step of determining aphysical property of the at least one RNA molecule having a cleavagesite by analyzing the at least one 5′ terminal RNA fragment or the 3′terminal RNA fragment and/or the at least one optional central RNAfragment obtained by cleaving the RNA molecule with the catalyticnucleic acid molecule into a 5′ terminal RNA fragment and at least one3′ RNA fragment and optionally into at least one central RNA fragment bycontacting the RNA molecule with the catalytic nucleic acid moleculeunder conditions allowing the cleavage of the RNA molecule.

Furthermore, it is particularly preferred that the present inventiondoes not relate to a method for determining the presence of a CAPstructure in an RNA molecule having a cleavage site for a catalyticnucleic acid molecule, a method for determining the capping degree of apopulation of RNA molecules having a cleavage site for a catalyticnucleic acid molecule, a method for determining the orientation of thecap structure in a capped RNA molecule having a cleavage site for acatalytic nucleic acid molecule and a method for determining relativeamounts of correctly capped RNA molecules and reverse-capped RNAmolecules in a population of RNA molecules, wherein the populationcomprises correctly capped and/or reverse-capped RNA molecules that havea cleavage site for a catalytic nucleic acid molecule.

In one embodiment the by-products comprise at least one nucleic acidmolecule, preferably at least two nucleic acid molecules with differentlength. This at least one nucleic acid molecule with different length ispresent in addition to the full-length target RNA so that in thedetection step by HPLC at least two peaks appear in the chromatogram,wherein the highest peak is the peak of the full-length target RNA andthe lower peak corresponds to the by-product which is present in thesample. The length of any short by-product is between 5 to 500nucleotides, preferably between 5 and 400 or between 5 and 300nucleotides, more preferably between 5 and 250 nucleotides and mostpreferably between 5 and 200 nucleotides. In particular, the by-productswhich are homooligomers of nucleotides have a length of 5 to 15nucleotides, preferably of 5 to 10 nucleotides, more preferably of 5 to7 nucleotides and most preferably of 5 nucleotides. The by-productswhich are short single-stranded RNAs produced by premature terminationof transcription have a length of 20 to 500 nucleotides, preferably of50 to 400 nucleotides, more preferably of 80 to 300 nucleotides and mostpreferably of 100 to 250 nucleotides. The length of the by-products canbe determined by HPLC, preferably by reversed-phase HPLC and morepreferably by ion-pair reversed-phase HPLC, wherein smaller moleculeselute earlier than larger molecules. An alternative method fordetermining the length of the by-products is capillary gelelectrophoresis.

The by-products of the present invention do not have a predeterminedsize, i.e. the size of the by-products only becomes apparent by themethod of the present invention and cannot be predicted based oncleavage sites present within the target RNA.

The target RNA is preferably longer than any of the by-products and mayhave a size of up to about 15000 nucleotides, preferably of 500 to 10000nucleotides, more preferably of 700 to 8000 nucleotides, even morepreferably 800 to 5000 nucleotides and most preferably 900 to 2000nucleotides.

The sample comprising the target RNA may be denatured before it ispurified according to step b) of the method of the present invention. Bydenaturing the sample comprising the target RNA any intramoleculardouble strands formed between two RNA strands or between an RNA strandand a DNA strand are disrupted so that in the following steps onlysingle-stranded nucleic acid molecules are present in the sample. Thesample comprising the target RNA may be denatured by heating the samplecomprising the in vitro transcribed RNA to a temperature at which thehydrogen bonds between the two strands are broken, such as a temperatureof 90° C. Alternatively or additionally, the sample comprising thetarget RNA may be treated with a denaturing agent such as urea.Preferably, within the method of the present invention the target RNA isnot treated with urea.

Purification of the Target RNA (Step b) of the Method of the Invention)

In step b) of the method of the present invention the target RNA ispurified by any suitable method. The method for purifying the target RNAis chosen so that the reagents (such as nucleotides and RNA polymerase)and by-products of the RNA in vitro transcription reaction are removedfrom the sample as completely as possible.

Suitable purification methods include alcoholic precipitation, LiClprecipitation, HPLC such as reversed-phase HPLC, anion exchangechromatography, hydroxyapatite chromatography and core beadchromatography, tangential flow filtration, gel filtrationchromatography, silica membranes such as, for example, PureYield™ RNAMidiprep System of Promega and affinity chromatography (either if a tagis attached to the target RNA or via the poly(A) stretch). Preferably,the target RNA is purified using reversed-phase HPLC. Also preferably,the step of purifying the target RNA does not involve a step of spinfiltration.

The HPLC for purifying the RNA is preferably performed on a preparativescale in which relatively large quantities of RNA are purified. Suchrelatively large quantities are for example quantities of 0.5 mg ormore, in particular 1.0 mg to 1000 mg or more, very particularlyapproximately 1.5 mg or more, upscaling even to the kg range beingpossible. The above statements are to be understood to mean that thesequantities relate to a single HPLC run. If a plurality of HPLC runs isperformed, the quantity increases in direct proportion to the number ofHPLC runs.

A particularly preferred method for purifying the target RNA isdisclosed in WO 2008/077592 A1 and involves a reversed-phase HPLC usinga porous reversed phase as stationary phase.

In general, any material known to be used as reverse phase stationaryphase, in particular any polymeric material may be used for theinventive method, if that material can be provided in porous form. Thestationary phase may be composed of organic and/or inorganic material.Examples for polymers to be used for the purification step of thepresent invention are (non-alkylated) polystyrenes, (non-alkylated)polystyrenedivinylbenzenes, silica gel, silica gel modified withnon-polar residues, particularly silica gel modified with alkylcontaining residues, more preferably with butyl-, octyl and/or octadecylcontaining residues, silica gel modified with phenylic residues,polymethacrylates, etc.

In a particularly preferred embodiment, the material for the reversedphase is a porous polystyrene polymer, a (non-alkylated) porouspolystyrenedivinylbenzene polymer, porous silica gel, porous silica gelmodified with non-polar residues, particularly porous silica gelmodified with alkyl containing residues, more preferably with butyl-,octyl and/or octadecyl containing residues, porous silica gel modifiedwith phenylic residues, porous polymethacrylates, wherein in particulara porous polystyrene polymer or a non-alkylated (porous)polystyrenedivinylbenzene may be used.

A non-alkylated porous polystyrenedivinylbenzene which is veryparticularly preferred for the purification step of the method accordingto the invention is one which, without being limited thereto, may have aparticle size of 8.0±1.5 μm, in particular 8.0±0.5 μm, and a pore sizeof 1000-1500 Å, in particular 1000-1200 Å or 3500-4500 Å.

The stationary phase is conventionally located in a column. V2A steel isconventionally used as the material for the column, but other materialsmay also be used for the column provided they are suitable for theconditions prevailing during HPLC. Conventionally the column isstraight. It is favourable for the HPLC column to have a length of 5 cmto 100 cm and a diameter of 4 mm to 25 mm. Columns used for thepurification step of the method of the invention may in particular havethe following dimensions: 50 mm long and 7.5 mm in diameter or 50 mmlong and 4.6 mm in diameter, or 50 mm long and 10 mm in diameter or anyother dimension with regard to length and diameter, which is suitablefor preparative recovery of RNA, even lengths of several metres and alsolarger diameters being feasible in the case of upscaling.

The HPLC is preferably performed as ion-pair, reversed phase HPLC asdefined above.

In a preferred embodiment, a mixture of an aqueous solvent and anorganic solvent is used as the mobile phase for eluting the RNA.Preferably, the buffer used as the aqueous solvent has a pH of 6.0-8.0,for example of about 7, for example 7.0. More preferably the buffer istriethylammonium acetate which preferably has a concentration of 0.02 Mto 0.5 M, more preferably of 0.08 M to 0.12 M. Most preferably, an 0.1 Mtriethylammonium acetate buffer is used, which also acts as a counterionto the RNA in the ion pair method.

In a preferred embodiment, the organic solvent which is used in themobile phase is selected from acetonitrile, methanol, ethanol,1-propanol, 2-propanol and acetone or a mixture thereof. More preferablyit is acetonitrile.

In a particularly preferred embodiment, the mobile phase is a mixture of0.1 M triethylammonium acetate, pH 7, and acetonitrile.

Preferably, the mobile phase contains 5.0 vol. % to 25.0 vol. % organicsolvent, relative to the mobile phase, and for this to be made up to 100vol. % with the aqueous solvent. Typically, in the event of gradientseparation, the proportion of organic solvent is increased, inparticular by at least 10%, more preferably by at least 50% and mostpreferably by at least 100%, optionally by at least 200%, relative tothe initial vol. % in the mobile phase. In a preferred embodiment, theproportion of organic solvent in the mobile phase amounts in the courseof HPLC separation to 3 to 9, preferably 4 to 7.5, in particular 5.0vol. %, in each case relative to the mobile phase. More preferably, theproportion of organic solvent in the mobile phase is increased in thecourse of HPLC separation from 3 to 9, in particular 5.0 vol. % to up to20.0 vol. %, in each case relative to the mobile phase. Still morepreferably, the method is performed in such a way that the proportion oforganic solvent in the mobile phase is increased in the course of HPLCseparation from 6.5 to 8.5, in particular 7.5 vol. %, to up to 17.5 vol.%, in each case relative to the mobile phase.

Even more preferably the mobile phase contains 7.5 vol. % to 17.5 vol. %organic solvent, relative to the mobile phase, and for this to be madeup to 100 vol. % with the aqueous buffered solvent.

Elution may proceed isocratically or by means of gradient separation. Inisocratic separation, elution of the RNA proceeds with a single eluentor a constant mixture of a plurality of eluents, wherein the solventsdescribed above in detail may be used as eluent.

In a preferred embodiment, gradient separation is performed wherein thecomposition of the eluent is varied by means of a gradient program. Theequipment necessary for gradient separation is known to a person skilledin the art. Gradient elution may here proceed either on the low pressureside by mixing chambers or on the high pressure side by further pumps.

Preferably, the proportion of organic solvent, as described above, isincreased relative to the aqueous solvent during gradient separation.The above-described agents may here be used as the aqueous solvent andthe likewise above-described agents may be used as the organic solvent.For example, the proportion of organic solvent in the mobile phase maybe increased in the course of HPLC separation from 5.0 vol. % to 20.0vol. %, in each case relative to the mobile phase. In particular, theproportion of organic solvent in the mobile phase may be increased inthe course of HPLC separation from 7.5 vol. % to 17.5 vol. %, inparticular 9.5 to 14.5 vol. %, in each case relative to the mobilephase.

The following gradient program has proven particularly favourable forthe purification of RNA:

Eluent A: 0.1 M triethylammonium acetate, pH 7Eluent B: 0.1 M triethylammonium acetate, pH 7, with 25 vol. %acetonitrile

Eluent Composition:

-   -   start: 62% A and 38% B (1 st to 3rd minute)    -   increase to 58% B (1.67% increase in B per minute), (3rd-15th        minute)    -   100% B (15th to 20th minute)

Another example of a gradient program is described below, the sameeluent A and B being used:

Eluent Composition:

-   -   starting level: 62% A and 38% B (1 st-3rd min)    -   separation range I: gradient 38%-49.5% B (5.75% increase in        B/min) (3rd-5th min)    -   separation range II: gradient 49.5%-57% B (0.83% increase in        B/min) (5th-14th min)    -   rinsing range: 100% B (15th-20th min)

It is preferred to use purified solvent for HPLC. Such purified solventsare commercially obtainable. They may additionally also be filteredthrough a 1 to 5 μm microfilter, which is generally mounted in thesystem upstream of the pump. It is additionally preferred for all thesolvents to be degassed prior to use, since otherwise gas bubbles occurin most pumps. If air bubbles occur in the solvent, they may interferenot only with separation but also with the continuous monitoring ofoutflow in the detector. The solvents may be degassed by heating, byvigorous stirring with a magnetic stirrer, by brief evacuation, byultrasonication or by passing a small stream of helium through thesolvent storage vessel.

The flow rate of the eluent is selected such that good separation of theRNA from the other constituents contained in the sample to beinvestigated takes place. The eluent flow rate may amount to from 1ml/min to several litres per minute (in the case of upscaling), inparticular about 1 to 1000 ml/min, more preferably 5 ml to 500 ml/min,even more preferably more than 100 ml/min, depending on the type andscope of the upscaling. This flow rate may be established and regulatedby the pump.

The HPLC is preferably performed under denaturing conditions, such as anincreased temperature. Suitable temperature conditions include atemperature of at least 70° C., preferably of at least 75° C., morepreferably of about 78° C. By using denaturing conditions anyintramolecular double strands formed between two RNA strands or betweenan RNA strand and a DNA strand are disrupted so that onlysingle-stranded nucleic acid molecules are present in the sample.

Detection proceeds preferably with a UV detector at 254 nm, wherein areference measurement may be made at 600 nm. However, any otherdetection method may alternatively be used, with which the RNA may bedetected.

For preparative purification of the RNA, it is advisable to collect theRNA-containing eluted solvent quantities. In this respect, it ispreferred to carry out this collection in such a way that the elutedsolvent is collected in individual separated fractions. This may takeplace for example with a fraction collector. In this way, thehigh-purity RNA-containing fractions may be separated from otherRNA-containing fractions which still contain undesired impurities,albeit in very small quantities. The individual fractions may becollected for example over 1 minute.

The method according to the invention is preferably performed undercompletely denaturing conditions. This may proceed for example in thatsample application takes place at a temperature of 4-12° C., the HPLCmethod otherwise proceeding at a higher temperature, preferably at 70°C. or more, particularly preferably at 75° C. or more, in particular upto 82° C., and very particularly preferably at about 78° C.

Sample application may be performed with two methods, stop-flowinjection or loop injection. For stop-flow injection a microsyringe isused which is able to withstand the high pressure applied in HPLC. Thesample is injected through a septum in an inlet valve either directlyonto the column packing or onto a small drop of inert materialimmediately over the packing. The system may in this case be underelevated pressure, or the pump may be turned off prior to injection,which is then performed when the pressure has fallen to close to thenormal value. In the case of loop injection, a loop injector is used tointroduce the sample. This consists of a tubular loop, into which thesample is inserted. By means of a suitable rotary valve, the stationaryphase is then conveyed out of the pump through the loop, whose outletleads directly into the column. The sample is entrained in this way bythe stationary phase into the column, without solvent flow to the pumpbeing interrupted.

Detection of by-Products (Step c) of the Method of the Invention)

After the target RNA has been purified to provide a purified target RNAsample, all or part of the purified target RNA sample is analyzed byHPLC to detect by-products. Preferably, only a part of the purifiedtarget RNA sample, such as 20% or 15%, preferably 10% or 8%, morepreferably 5% or 2% and most preferably 1% of the volume of the purifiedtarget RNA sample or less is used for the HPLC analysis to detectby-products.

The HPLC analysis of step c) of the method of the present invention istherefore preferably performed at an analytical scale. In an analyticalHPLC method, a quantity of RNA such as 8 ng to 1000 ng or 20 to 100 μgis introduced for a single HPLC run. If a plurality of HPLC runs isperformed, the quantity increases in direct proportion to the number ofHPLC runs.

The remainder of the purified target RNA sample can be further processedto the final RNA product, such as a RNA product for administration to apatient, if the HPLC analysis according to step c) of the method of theinvention indicates that the amount of by-products is within a rangewhich is acceptable for a final RNA product.

Stationary phases for use in the HPLC analysis are known in the art.Preferably, the stationary phase is selected from the group consistingof a porous polystyrene, a porous non-alkylated polystyrene, apolystyrenedivinylbenzene, a porous non-alkylatedpolystyrenedivinylbenzene, a porous silica gel, a porous silica gelmodified with non-polar residues, a porous silica gel modified withcarbon chains, selected from butyl-, octyl and/or octadecyl carbonchains, a porous silica gel modified with phenylic residues, and aporous polymethacrylate. The stationary phase used for step c) of themethod of the present invention is preferably a porous silica gelmodified with carbon chains, preferably an octadecyl carbon chain.

More preferably the porous silica gel is prepared from tetraethoxysilaneand bis(triethoxysilyl)ethane which are even more preferably used in a4:1 mole ratio. Most preferably, the porous silica gel is prepared fromtetraethoxysilane and bis(triethoxysilyl)ethane which are used in a 4:1mole ratio and the porous silica gel is modified with an octadecylcarbon chain. Such porous silica gel is commercially available, e.g.XBRIDGE™ OST C₁₈ from Waters or AQUITY UPLC OST C18 from Waters, and isdescribed in more detail in Wyndham et al. (2003) Anal. Chem.75(24):6781-8 and WO 2003/014450.

The silica gel may have a particle size of 0.5 to 5 μm, preferably of0.7 to 4 μm, more preferably of 1 to 3 μm, even more preferably of 1.5to 2 μm and most preferably of 1.7 μm. The pore size of the poroussilica gel may be 50 to 300 Å, preferably 70 to 250 Å, more preferably100 to 200 Å, even more preferably 120 to 170 Å and most preferably itis 135 Å.

Hence, in a most preferred embodiment the stationary phase is a poroussilica gel prepared from tetraethoxysilane and bis(triethoxysilyl)ethanewhich are used in a 4:1 mole ratio, wherein the porous silica gel ismodified with an octadecyl carbon chain and has a pore size of 135 Å anda particle size of 1.7 μm.

The stationary phase is conventionally located in a column. V2A steel isconventionally used as the material for the column, but other materialsmay also be used for the column provided they are suitable for theconditions prevailing during HPLC. Conventionally the column isstraight. It is preferred for the HPLC column to have a length of 5 cmto 100 cm and a diameter of 0.5 mm to 10 mm. Columns used for the methodaccording to the invention may in particular have the followingdimensions: 50 mm long and 4.6 mm in diameter or 50 mm long and 2.1 mmin diameter.

The HPLC is preferably performed as ion-pair, reversed phase HPLC asdefined above.

In a preferred embodiment, a mixture of an aqueous solvent, preferably abuffer, and an organic solvent is used as the mobile phase for elutingthe RNA.

Preferably, the buffer used as the aqueous solvent has a pH of 6.0-8.0,for example of about 7, for example 7.0.

The buffer may be selected from the group consisting of triethylammoniumacetate, trifluoroacetic acid, acetic acid, formic acid, acetate buffer,phosphate buffer, tetrabutylammonium bisulfate, tetrabutylammoniumbromide and tetrabutylammonium chloride.

Preferably the buffer is triethylammonium acetate which preferably has aconcentration of 0.02 M to 0.5 M, more preferably of 0.08 M to 0.12 M.Most preferably, an about 0.1 M triethylammonium acetate buffer is used,which also acts as a counterion to the RNA in the ion pair method.

In a preferred embodiment, the organic solvent which is used in themobile phase is selected from acetonitrile, methanol, ethanol,1-propanol, 2-propanol, hexafluoroisopropanol and acetone or a mixturethereof. More preferably it is acetonitrile.

In a particularly preferred embodiment, the mobile phase is a mixture of0.1 M triethylammonium acetate, pH 7.3, and acetonitrile.

Preferably, the mobile phase which is applied to the HPLC columncontains 3.0 vol. % to 5.0 vol. % organic solvent, preferably 3.25 to4.0 vol. % and most preferably 3.75 vol. % organic solvent, preferablyacetonitrile, relative to the mobile phase, and is made up to 100 vol. %with the aqueous solvent.

Elution may proceed isocratically or by means of gradient separation. Inisocratic separation, elution of the RNA proceeds with a single eluentor a constant mixture of a plurality of eluents, wherein the solventsdescribed above in detail may be used as eluent. In a preferredembodiment, gradient separation is performed wherein the composition ofthe eluent is varied by means of a gradient program. The equipmentnecessary for gradient separation is known to a person skilled in theart. Gradient elution may here proceed either on the low pressure sideby mixing chambers or on the high pressure side by further pumps.

Typically, in the event of gradient separation, the proportion oforganic solvent, in particular of acetonitrile, is increased in thecourse of HPLC separation in one or more steps. Preferably, theproportion of the organic solvent, in particular of acetonitrile, isincreased in the course of HPLC separation in two steps, more preferablythe proportion of the organic solvent, in particular of acetonitrile, isincreased in the course of HPLC separation in three steps.

In one embodiment, the proportion of the organic solvent, in particularof acetonitrile, is increased in the course of HPLC separation from 3.5vol. % to 100 vol. %, relative to the mobile phase, and is made up to100 vol. % with the aqueous solvent. After increasing the proportion ofthe organic solvent, in particular of acetonitrile, the proportion mayagain be decreased, e.g. from 100 vol. % to 3.5 vol. %, the rest beingthe aqueous solvent.

The following gradient program has proven particularly useful in theHPLC method for detecting by-products:

Eluent A: 0.1 M triethylammonium acetate, pH 6.8Eluent B: 0.1 M triethylammonium acetate, pH 7.3, with 25 vol. %acetonitrile

Eluent Composition:

-   -   start: 86% A and 14% B (1 st to 3rd minute)    -   increase to 19% B over 2 minutes    -   increase to 21% B over 9 minutes, then holding at 21% B for one        minute    -   increase to 100% B over 5 minutes, then holding at 100% B for        3.5 minutes    -   decrease to 14% over 1.5 minutes

It is preferred to use purified solvent for HPLC. Such purified solventsare commercially obtainable. They may additionally also be filteredthrough a 1 to 5 μm microfilter, which is generally mounted in thesystem upstream of the pump. It is additionally preferred for all thesolvents to be degassed prior to use, since otherwise gas bubbles occurin most pumps. If air bubbles occur in the solvent, they may interferenot only with separation but also with the continuous monitoring ofoutflow in the detector. The solvents may be degassed by heating, byvigorous stirring with a magnetic stirrer, by brief evacuation, byultrasonication or by passing a small stream of helium through thesolvent storage vessel.

The flow rate of the eluent is selected such that good separation of theRNA from the other constituents contained in the sample to beinvestigated takes place. The eluent flow rate is between 50 and 80ml/min, preferably between 55 and 75 ml/min, more preferably between 60and 70 ml/min and most preferably it is 0.65 ml/min. This flow rate maybe established and regulated by the pump.

Detection proceeds preferably with a UV detector at 254 nm, wherein areference measurement may be made at 600 nm. However, any otherdetection method may alternatively be used, with which the RNA may bedetected.

The method according to the invention is preferably performed atelevated temperature. For example, the sample comprising the purifiedtarget RNA or a fraction thereof may be applied to the column at atemperature of 4-12° C., and the subsequent steps may be performed at ahigher temperature, preferably at 50° C. or more, particularlypreferably at 55° C. or more and most preferably at about 60° C.

Sample application may be performed with two methods, stop-flowinjection or loop injection. For stop-flow injection a microsyringe isused which is able to withstand the high pressure applied in HPLC. Thesample is injected through a septum in an inlet valve either directlyonto the column packing or onto a small drop of inert materialimmediately over the packing. The system may in this case be underelevated pressure, or the pump may be turned off prior to injection,which is then performed when the pressure has fallen to close to thenormal value. In the case of loop injection, a loop injector is used tointroduce the sample. This consists of a tubular loop, into which thesample is inserted. By means of a suitable rotary valve, the stationaryphase is then conveyed out of the pump through the loop, whose outletleads directly into the column. The sample is entrained in this way bythe stationary phase into the column, without solvent flow to the pumpbeing interrupted.

Within the method of the present invention the step of detectingby-products may also comprise determining the amount of each by-productor the total amount of all by-products present in the sample.

The term “amount of each by-product”, as used herein, means the amountof a specific by-product present within a sample. It can be determinedby calculating the area of the peak corresponding to said specificby-product and relating this area to the area of the peak of the targetRNA.

The term “total amount of all by-products”, as used herein, means thetotal amount of all by-products present within a sample. It can bedetermined by calculating the area of all peaks representing by-productsand relating this area to the area of the peak of the target RNA.

After detecting the by-products in the purified target RNA sample byHPLC, said by-products may be isolated and/or characterized. Theby-products may be isolated by collecting the RNA-containing elutedsolvent quantities. In this respect, it is preferred to carry out thiscollection in such a way that the eluted solvent is collected inindividual separated fractions. This may take place for example with afraction collector. In this way, the by-product-containing fractions maybe separated from the fractions containing the target RNA. Further, ifthe HPLC analysis shows more than one by-product peak, each of theby-products corresponding to one of the peaks may be collectedseparately, allowing the separate analysis of each by-product. Theindividual fractions may be collected for example over 1 minute.

The by-products may be characterized by any suitable method of RNAanalysis, including enzyme assays, spectroscopic methods, massspectrometry and sequencing.

Spectroscopic methods for RNA analysis include traditional absorbancemeasurements at 260 nm and more sensitive fluorescence techniques usingfluorescent dyes such as ethidium bromide and a fluorometer with anexcitation wavelength of 302 or 546 nm (Gallagher (2011) CurrentProtocols in Molecular Biology. 93:A.3D.1-A.3D.14).

A mass spectrometer (MS) is a gas phase spectrometer that measures aparameter that can be translated into mass-to-charge ratio of gas phaseions. Examples of mass spectrometers are time-of-flight, magneticsector, quadrupole filter, ion trap, ion cyclotron resonance,electrostatic sector analyser and hybrids of these. Methods for theapplication of MS methods to the characterization of nucleic acids areknown in the art.

For example, Matrix-Assisted Laser Desorption/Ionization MassSpectrometry (MALDI-MS) can be used to analyse oligonucleotides at the120-mer level and below (Castleberry et al. (2008) Current Protocols inNucleic Acid Chemistry. 33: 10.1.1-10.1.21).

Electrospray Ionization Mass Spectrometry (ESI-MS) allows the analysisof high-molecular-weight compounds through the generation of multiplycharged ions in the gas phase and can be applied to molecular weightdetermination, sequencing and analysis of oligonucleotide mixtures(Castleberry et al. (2008) Current Protocols in Nucleic Acid Chemistry.35: 10.2.1-10.2.19). Preferably, the mass spectrometry analysis isconducted in a quantitative manner to determine the amount of RNA.

Methods for sequencing of RNA are known in the art. A recently developedtechnique called RNA Sequencing (RNA-Seq) uses massively parallelsequencing to allow for example transcriptome analyses of genomes at afar higher resolution than is available with Sanger sequencing andmicroarray-based methods. In the RNA-Seq method, complementary DNAs(cDNAs) generated from the RNA of interest are directly sequenced usingnext-generation sequencing technologies. RNA-Seq has been usedsuccessfully to precisely quantify transcript levels, confirm or revisepreviously annotated 5′ and 3′ ends of genes, and map exon/intronboundaries (Eminaga et al. (2013) Current Protocols in MolecularBiology. 103: 4.17.1-4.17.14). Consequently, the amount of theby-products can be determined also by RNA sequencing.

The method of the present invention may be used to identify sequencemotifs within the RNA which are responsible for the generation of theby-products. For example, several RNA sequences encoding the sameprotein, but differing in the coding sequence or the presence and/ortype of 5′ or 3′ untranslated region may be produced and the amount andoptionally the identity of the by-products can be determined for each ofthese RNA sequences by the method of the present invention, leading tothe selection of a construct encoding the RNA product which constructproduces the lowest amount of by-products.

The method of the present invention may also be used for the qualitycontrol of RNA produced by RNA in vitro transcription. For example, inthe preparation of RNA products for pharmaceutical use a level ofby-products may be defined which is acceptable for the pharmaceuticalproduct. The method of the present invention can then be used todetermine whether in a sample the total amount of the by-products isbelow or above the threshold. If the amount of by-products is below thethreshold, the RNA product can be marketed and if the amount of theby-products is above the threshold, the RNA product has to be discardedor subjected to further purification steps.

Further, the method of the present invention may be used to comparedifferent RNA purification conditions. For example, one or morevariables in a purification protocol can be varied and then it can bedetermined whether the amount of by-products increases or decreases dueto the variation of the purification conditions. Then those purificationconditions which produce the lowest amount of by-products are selectedfor the preparative RNA purification.

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

Example 1: Preparation of the mRNA 1. Preparation of DNA Template

For the present example DNA sequences encoding PpLuc mRNA according toSEQ ID NOs: 1-3 were prepared and used for subsequent in vitrotranscription reactions. The RNAs encoded by the DNA sequences had thefollowing features:

-   -   5′ cap—GC-optimized open reading frame (ORF)—globin 3′ UTR—a        stretch of 64 adenosines—a stretch of 30 cytosines (RNA R 491)    -   5′ cap—GC-optimized open reading frame (ORF)—globin 3′ UTR—a        stretch of 64 adenosines—a stretch of 30 cytosines—a histone        stem-loop sequence (RNA R 1265)    -   5′ cap—32L-5′-UTR—GC-optimized open reading frame (ORF)—albumin        3′ UTR—a stretch of 64 adenosines—a stretch of 30 cytosines—a        histone stem-loop sequence (RNA R 2244)

The constructs were prepared by modifying the wild type coding sequenceby introducing a GC-optimized sequence for stabilization, UTRs (derivedfrom 32L4, albumin or alpha globin were introduced as indicated). The3′-UTR was followed by a stretch of 64 adenosines (poly-A-sequence), astretch of 30 cytosines (poly-C-sequence) and optionally a histonestem-loop sequence.

2. In Vitro Transcription

The DNA plasmids prepared according to section 1 were transcribed invitro using T7 RNA polymerase.

For the production of 5′-capped RNAs using cap analog, transcription wascarried out in 5.8 mM m7G(5″)ppp(5′)G Cap analog, 4 mM ATP, 4 mM CTP, 4mM UTP, and 1.45 mM GTP (all Thermo Fisher Scientific). Subsequently themRNA was purified using HPLC using a porous reversed phase as stationaryphase (described in detail in WO2008/077592A1).

Example 2: HPLC Determination of Short RNA by-Products

Analysis was performed via ion-pair, reversed-phase chromatography on aDionex Parallel-HPLC U3000 CV-P-1247, equipped with analytical pump(DPG-3600SD), column oven (TCC-3000SD) and UV/Vis-4-channel-detectors(2×VWD-3400RS) with analytical SST measuring cell (11 μL, 10 mm, forVWD-3×00 detector). An AQUITY UPLC OST C18 column (2.1×50 mm, 1.7 μmparticle size; Waters Corporation, Milford, Mass., USA) was used. Columntemperature was set to 60° C. Buffer A contained 0.1 M triethylammoniumacetate (TEAA), pH 6.8, buffer B 0.1 M TEAA, pH 7.3, 25% acetonitrile.The column was equilibrated with 14% buffer B.

For sample preparation, HPLC equilibration buffer (86% buffer A, 14%buffer B) was added to the RNA to obtain a final volume of 1700 μl.

1650 μl of the RNA solution were loaded using a SEMIPREP-Autosampler(WPS-3000SL, Dionex) and run with a stepped gradient beginning with 14%buffer B for 3 minutes, increasing to 19% buffer B over 2 minutes, to21% buffer B over 9 minutes. 21% buffer B was held for 1 minute, thenincreased to 100% B over 5 minutes, held for 3.5 minutes, then decreasedto 14% buffer B over 1.5 minutes.

Signal integration was done using Chromeleon software 6.80 SR11 Build3161 (Dionex).

FIG. 1 shows that by applying a standard preparative HPLC method severalpeaks representing by-products of the RNA in vitro transcriptionreaction can be detected.

FIG. 2 shows that by applying an improved preparative HPLC methodshorter by-products (shown as peaks (1) and (2) in FIG. 1B) could beremoved, thereby increasing the quality of the mRNA product.

1. Method for detecting by-products of in vitro transcription in a sample comprising an in vitro transcribed target RNA, the method comprising the steps of: a) preparing a sample comprising a target RNA by in vitro transcription; b) purifying the target RNA, thereby providing a purified target RNA sample; c) detecting the by-products in the purified target RNA sample by HPLC.
 2. Method according to claim 1, wherein the method does not comprise a step of treating the target RNA with a ribozyme.
 3. Method according to claim 1 or 2, wherein the by-products comprise at least two nucleic acid molecules with different length.
 4. Method according to any one of the preceding claims, wherein the by-products do not comprise the 3′ terminus of the target RNA.
 5. Method according to any one of the preceding claims, wherein the by-products have a length of 5 to 500 nucleotides.
 6. Method according to any one of the preceding claims, wherein the by-products are homooligomers of nucleotides, short single-stranded RNAs, double-stranded RNAs and/or DNA-RNA hybrids.
 7. Method according to any one of the preceding claims, wherein step b) is performed under denaturing conditions.
 8. Method according to any one of the preceding claims, wherein step b) comprises a step of purifying the target RNA by HPLC.
 9. Method according to any one of the preceding claims, wherein step b) comprises a step of purifying the target RNA by reversed-phase HPLC.
 10. Method according to claim 8 or 9, wherein a porous reversed phase is used as stationary phase in the HPLC.
 11. Method according to claim 10, wherein the porous reversed phase is a porous, non-alkylated polystyrene/divinylbenzene matrix.
 12. Method according to any one of the preceding claims, wherein the HPLC in step c) is ion-pair, reversed-phase HPLC.
 13. Method according to any one of the preceding claims, wherein the HPLC in step c) uses a carbon-chain bonded silica column.
 14. Method according to claim 13, wherein the carbon-chain bonded silica column is an octadecyl carbon chain (C18)-bonded silica column.
 15. Method according to claim 13 or 14, wherein the silica column is prepared from tetraethoxysilane and bis(triethoxysilyl)ethane.
 16. Method according to claim 15, wherein tetraethoxysilane and bis(triethoxysilyl)ethane are used in a 4:1 mole ratio.
 17. Method according to any one of claims 13 to 16, wherein the column has a particle size of 0.5 to 5 μm.
 18. Method according to any one of claims 13 to 17, wherein the column has a pore size of 50 to 300 Å.
 19. Method according to any one of the preceding claims, wherein the HPLC of step c) uses a mixture of an aqueous solvent and an organic solvent as mobile phase.
 20. Method according to claim 19, wherein the aqueous solvent is a buffer.
 21. Method according to claim 20, wherein the buffer is selected from the group consisting of triethylammonium acetate, trifluoroacetic acid, acetic acid, formic acid, acetate buffer, phosphate buffer, tetrabutylammonium bisulfate, tetrabutylammonium bromide and tetrabutylammonium chloride.
 22. Method according to claim 20 or 21, wherein the buffer is a 0.1 M triethylammonium acetate buffer.
 23. Method according to any one of claims 19 to 22, wherein the organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, 1-propanol, 2-propanol, hexafluoroisopropanol, acetone and a mixture thereof.
 24. Method according to any one of claims 19 to 23, wherein the organic solvent is acetonitrile.
 25. Method according to any one of claims 19 to 24, wherein at the beginning of the HPLC process the mobile phase contains 3 to 5% organic solvent, relative to the mobile phase, the rest being the aqueous solvent.
 26. Method according to any one of claims 12 to 25, wherein a gradient separation proceeds.
 27. Method according to claim 26, wherein the proportion of organic solvent is increased to provide the gradient.
 28. Method according to claim 27, wherein the proportion of organic solvent in the mobile phase is increased in the course of HPLC separation from 3.5% to 100%.
 29. Method according to any one of the preceding claims, wherein the method further comprises a step d) of isolating and/or characterizing the by-products.
 30. Method according to claim 29, wherein the by-products are characterized by enzyme assays, mass spectrometry and/or sequencing.
 31. Method according to any one of the preceding claims, wherein the amount of the by-products relative to the total amount of RNA is determined.
 32. Use of the method according to any one of the preceding claims for identifying sequence motifs within the target RNA which are responsible for the generation of by-products.
 33. Use of the method according to any one of the preceding claims for the quality control of RNA produced by in vitro transcription.
 34. Use of the method according to any one of claims 1 to 31 for identifying suitable RNA purification conditions.
 35. Use of the method according to any one of claims 1 to 31 for comparing RNA purification conditions. 